全 文 ：Phylogenetic Relationships of Caragana
(Fabaceae) by the Use of nrITS Sequences
Hongmei KANG1,2, Juan BAI3, Kang CHEN4, Gang WANG1*
1. Key Laboratory of Arid and Grassland Agroecology of Ministry of Education, School of life Sciences, Lanzhou University,
Lanzhou 730000, China;
2. Institute of Biology, Gansu Academy of Sciences, Lanzhou 730000, China;
3. College of Life Science, Northwest A & F University, Xi’an 712100, China;
4. Institute of Oceanology, Chinese Academy of Science, Qingdao 266071, China
Supported by National Key Basic Research Development Program of China
(2002CB111505).
*Corresponding author. E-mail: wangg@lzu.edu.cn
Received: November 2, 2011 Accepted: November 28, 2011
The paper was from Southwest China Journal of Agriculture Sciences 2011 24(3).
(China).A
Abstract [Objective] The aim was to explore the phylogenetic relationships of Cara-
gana (Fabaceae) by the use of nrITS sequences. [Method] Internal transcribed spac-
er (ITS) sequences of nuclear ribosomal DNA (nrDNA) from 29 taxa of Caragana
species and seven close relatives (all belong to Astralinae (Adens) Benth) were
used for phylogenetic analysis. [Result] Length of the entire ITS region ranges from
611 to 614 bp in Caragana species. The aligned sequences nrITS of Caragana are
655 bp, and 170 sites are variable, with 107 phylogenetically informative sites. The
phylogenetically informative sites are 16.3% of the total aligned sequences. The ITS
sequences data are useful to resolve some relationships at lower taxonomic levels
within Caragana. The Caragana Fabr. is not a monophyletic group with very close
connection with Calophaca tianschanica. The ITS data revealed that the species of
Sect. tragacanthoides were dispersed in MP tree or ME tree. Although the morphol-
ogy of C. ordosica is similar to C. tibetica, the ITS results revealed an unexpectedly
close relativeship to C. roborovskyi. The ITS data also indicate C. davazamcii, C.
korshinskii, C. intermedia, and C. microphylla are different species. [Conclusion] ITS
sequences have an important reference value in exploring the relationships of Cara-
gana.
Key words Caragana Fabr.; ITS sequence; Phylogenetic relationships
C aragana Fabr. belongs to Pa-pilionoideae of Leguminosae.In the worldwide, there are
about 70 species of Caragana Fabr. [1],
which are widely distributed in temper-
ate regions of Asia[2]. In China, there are
62 kinds, 9 variants and 12 geographic
variants[3], which are mainly distributed
in the steppe and desert areas. These
species have strong resistance to
drought, cold and barren, thus having
abilities of sand-fixing and soil and wa-
ter conservation, as well as a wide
range of applications. Therefore, these
species have been paid more and
more attentions.
Caragana Fabr. is established by
PJ Fabricius in 1 763 based on the
model of Robinia Caragana L. in “Spe-
cies Plntarum” published by Linnaeus
in 1 753[1]. Since then, there have been
many researches on the classification
of Caragana Fabr., and the results are
different. In 1908, Komarov recorded
eight lines and 55 species in this
genus, which were considered as ba-
sis for the further study. Sanczir pro-
posed a more complete classification
system for Caragana Fabr. in 1979,
which recorded three groups, 15 lines
and 92 species. As for 1993, this genus
was classified to 11 lines by LIU Ying-
xin in Flora of China. In the same
year, this genus was classified to three
subgenera, five groups and 10 lines by
ZHAO Yi-zhi[4]. Based on the morpho-
logical, chromosomological and paly-
nological characters, ZHANG Ming-li [5]
carried out studies on the phylognetic
relationships of Caragana Fabr. using
cladistic systematics. The result
showed that 72 species in this genus
should be classified into 12 lines and
five groups. According to previous
studies and amendments, ZHOU Dao-
wei[6] thought that there were about 80
species worldwide, which can be divid-
ed into six groups, 14 lines.
Because of a great variety of
species inCaragana Fabr., complicated
intraspecies variations, convergent ev-
olution, hybridization and other factors
that blurred the interspecies bound-
aries, the classification of this genus
was difficult and has caused some
controversies, such as the taxonomic
status of C. davazamcii Sancz and the
relationship among it and C. korshin-
skii [7], C. intermedia and C. korshinskii,
as well as the reasonability of three
subgenera defined by ZHAO Yi-zhi [3].
ITS sequence has been widely used in
the analysis of phylogenetic relation-
ship among plant genus and related
genera because of its moderate evolu-
tion rate and easy amplification[8-9]. In or-
der to fully develop and utilize the
Caragana Fabr. resources, the use of
molecular biology techniques com-
bined with morphological principles for
the studies on the molecular system-
atics not only has important scientific
significance, but also has very impor-
tant economic significance.
Materials and Methods
Materials
The experimental species were
collected from Sand Plant Garden of
Gansu Desert Control Research Insti-
tute (Minqin, Gansu) and Shapotou
Workstation Introduction Garden of
Agricultural Science & Technology, 2012, 13(1): 36-39
Copyright訫 2012, Information Institute of HAAS. All rights reserved Agricultural Biotechnology
DOI:10.16175/j.cnki.1009-4229.2012.01.047
Agricultural Science & Technology2012
Vol.13, No.1, 2012 Agricultural Science & Technology
Cold and Arid Regions Environmental
and Engineering Research Institute,
Chinese Academy of Sciences (Zho-
ngwei, Ningxia). The other sequences
were searched from genBank. The
species name, accession number,
distribution were shown in Table 1.
Method
Total DNA was extracted using
CTAB method[11]. rDNA ITS fragments
were amplified by P1 + P4 (including
ITS-1, 5.8S rDNA and ITS-2)[12]. Reac-
tion system was 50 μl, including: 50 ng
DNA, 200 μmol/L dNTP, 0.2 μmol/L of
each primer, 1 U Pfu polymerase, 5 μl
10 × Pfu buffer. Reaction procedures
were: 94 ℃ denaturation for 3 min; 94
℃ denaturation for 1 min, 55 ℃ an-
nealing for 1 min, 72℃ extension for 3
min, 35 cycles; 72 ℃ extension for 7
min.
PCR products were purified and
bi-directional sequenced by Sangon
Biotech (Shanghai) Co., Ltd.
The range of ITS sequences was
determined according to C. arboresce-
ns in GenBank[13]. Sequence alignment
was performed using ClustalX 1.81
software[14]. The phylogenetic relation-
ship was analyzed by MEGA 3.1 [15].
The maximum parsimony (MP) and
minimum evolution (ME) methods
were used for cluster analysis, and the
Bootstrap test (500 repetitions) was
carried out in order to further deter-
mine the confidence of each branch.
Table 1 List of the materials used for molecular analysis, together with their geographic distribution and accession numbers
Speciesa Distributionb Number
Ser. Dasyphyllae Pojark
C. dasyphylla Pojark Xinjiang, the Former Soviet Union DQ914786
Ser. Chamlagu Pojark.
C. sinica (Buchoz) Rehd. Southwestern, northwestern and most parts of China DQ914785
Ser. Spinosae Kom.
C. pruinosa Kom. Xinjiang, the Former Soviet Union AB262533c
C. erinacea Kom. Gansu, Tibet, Sichuan, Qinghai, Yunnan AB262532c
C. tragacanthoides Poir. Xinjiang, the Former Soviet Union AB262536c
Ser. Occidentales Kom.
C. acanthophylla Pojark. Xinjiang, the Former Soviet Union AB262523c
C. bicolor Kom. Sichuan, Yunnan, Tibet DQ914790
Ser. Jubatae Kom.
C. jubate (Pall.) Poir. Inner Mongolia, Hebei, Shanxi, Xinjiang DQ914787
Ser. Tragacanthoides Pojark.
C. hololeuca Bunge ex Kom. Xinjiang, the Former Soviet Union AB262524c
C. roborovskyi Kom. Inner Mongolia, Xinjiang, Shaanxi, Ningxia, Qinghai, Gansu, Tibet AF521958
C. bongardiana Pojark. Xinjiang, the Former Soviet Union AB262528c
C. tibetica Kom Gansu, Qinghai, Xinjiang, Ningxia AB262527c
Inner Mongolia, Sichuan, Tibet
C. ordosica Y.Z. Zhao Mongolia, Inner Mongolia, Ningxia, Gansu DQ914780
Ser. Caragana
C. zahlbruckneri Schneid Beijing, Hebei, Shanxi, Inner Mongolia, Heilongjiang AB262530c
C. arborescens Lam. Shanxi, Shaanxi, Hebei, Shandong, Liaoning, Heilongjiang, Inner Mongolia, DQ311963
Xinjiang, Gansu, the former Soviet Union, Mongolia
C. sibirica Fabr. the former Soviet Union AY626912
Ser. Microphyllae Kom.
C. davazameii Sancz. Inner Mongolia AY626913
C. microphylla Lam. Shanxi, Shaanxi, Inner Mongolia, Xinjiang, Gansu, Ningxia, Shandong, DQ311964
Hebei, Sichuan, Jilin, Heilongjiang, Liaoning, Anhui, Tibet, Japan,
Mongolia, Russia
C. intermedia Kuang et H.C.Fu Shanxi, Shaanxi, Ningxia, Inner Mongolia AB262534c
C. korshinskii Kom. Gansu, Ningxia, Inner Mongolia, Shanxi, Shanxi, Mongolia AY626914
C. purdomii Rehd. Shanxi, Shaanxi, Inner Mongolia AB262529c
Ser. Pygmaeae Kom.
C. leucophloea Pojark. Gansu, Inner Mongolia, Xinjiang, the former Soviet Union, Mongolia AB262535c
C. stenophylla Pojark. Northeast China, Hebei, Shanxi, Inner Mongolia, Gansu, Shaanxi AB262525c
Ningxia, Russia, Mongolia
Ser. Grandiflorae Pojark.
C. brachypoda Pojark. Ningxia, Gansu, Inner Mongolia, Mongolia DQ914783
C. opulens Kom. Inner Mongolia, Hebei, Ningxia, Shaanxi, Gansu, Qinghai, AB262531c
Tibet, Sichuan
Ser. Frutescentes Pojark.
C. frutex (Linn.) C. Koch Henan, Hebei, Shandong, Shanxi, Jiangsu U56001, U56002
Shaanxi, Gansu, Xinjiang
C. rosea Turcz. Ex Maxim. Northeast China, Hebei, Inner Mongolia, Shaanxi, Shanxi, Henan, Gansu AB262526c
Jiangsu, Zhejiang, Anhui, Sichuan, Japan, Russian
C. licentiana Hand.-Mazz. Shanxi, Xinjiang, Sichuan, the former Soviet Union DQ914776
C. densa Kom. Sichuan, Qinghai, Gansu, Xinjiang DQ914788
a: Classification of Caragana is according to LIU Ying-xin; b: Distributions are from NIU Xi-wu[10]; c: The nucleotide data sequenced by Key
Laboratory of Arid and Grassland Agroecology of Ministry of Education, School of life Sciences, Lanzhou University.
37
Agricultural Science & Technology 2012
Agricultural Science & Technology Vol.13, No.1, 2012
■Sect. Prutnase; ●Sect. Longispina; ▲ Sect. Frutescentes; ◇Sect. Tragacanthoides;
△Sect. Caragana.
Fig.1 One of the 81 equally most parsimonious trees that is topologically identical to the
50% majority-rule consensus tree of 29 Caragana and related genus, based on phy-
logenetic analysis of ITS sequence data (CI = 0.751 572; RI = 0.795 337).
Results and Analysis
In this study and the previous
works, the nrITS sequences of 11
lines, 29 species of Caragana Fabr.
(including ITS-1, 5.8S rDNA, ITS-2)
were sequenced. The entire length of
ITS sequence of Caragana Fabr.
plants ranged from 611 to 614 bp; with
the ITS-1 sequence length of 229 to
231 bp, 5.8S sequence length of 163
bp and ITS-2 sequence length of 216
bp to 220 bp. The G + C content within
the ITS-1 region ranged from 55.6% to
60.4% (with an average of 58.3% ),
while that of ITS-2 region was of
48.4% to 50.7% (with an average of
49.4%). The aligned sequences nrITS
of Caragana are 655 bp, and 170 sites
are variable, with 107 phylogenetically
informative sites. The phylogenetically
informative sites are 16.3% of the total
aligned sequences, indicating higher
information content, and therefore
having higher value in exploring the
phylogenitic relationships among
genus and relatives of Caragana.
According to sequencing results,
the ITS-1, 5.8S rDNA and ITS-2 were
considered as a common matrix for
parsimony analysis and minimum
evolution analysis. A total of 81 MP
trees were generated, and MP trees
were shown in Fig.1. 29 species of
Caragana were clustered into five
branches. ME trees (data not shown)
were similar to MP trees. Caragana
Fabr. was not a monophyletic group.
Discussion and Conclusion
ITS phylogenetic analysis showed
that Caragana Fabr. was not a mono-
phyletic group. Caloplaca tianschanica
and C. bicolor were clustered into one
branch (BV=89%). Calophaca Fish.ex
DC. was a small genus belonging to
Trib.Galegeae and was related to
Caragana Fabr, Chesneya Lind1.ex
End1. Most of the species in Calopha-
ca Fish.ex DC. were distributed in the
mountains of Central Asia . The results
in this study suggested that the plants
in Caragana Fabr. were closely related
to that in Calophaca Fish.ex DC.
Caragana Fabr. and Calophaca Fish.
ex DC. are both of discontinuous dis-
tribution, and the study on them has
great significance on researches on
the origin and development of Asian
inland xeric flora[16].
For the classification of plants in
Caragana Fabr., the existed studies
are based on the morpholog evidence.
ZHAO Yi-zhi classified this genus into
three subgenus according to the rachis
abscission or persistent rachis, namely
rachis abscission subgenus, persistent
subgenus and persistent rachis ab-
scission subgenus. HOU Xin et al. [17]
detected and analyzed the ITS, trnL-F
and trnS-G sequence of 20 represen-
tative species of the Caragana Fabr.,
and the results were consistent with
that of ZHAO Yi-zhi. In this study, the
phylogenetic trees constructed by the
ITS sequences of 29 species of Cara-
gana Fabr. did not cluster to three dis-
tinct branches, indicating that the
studies on the phylogenetic study of
Caragana Fabr. based on only a few
representative species was not e-
nough.
Based on the previous studies,
ZHOU Dao-wei classified the Cara-
gana Fabr. plants into six groups, 13
lines, in which Sect. Tragacanthoides
Sancz. consisted of three lines,
namely, Ser. Laetevirentes, Ser. Eri-
nacanthae and Ser. Jubatae. In this
study, four species in Sect. Tragacan-
thoides were gathered in the Clade A
and Clade D, suggesting that the clas-
sification of this group needed further
study.
C. ordosica was a new species
published in 2005 by ZHAO Yi-Zhi et
al. [18], distributing in Mongolia, Inner
Mongolia, Ningxia and Gansu. This
species was similar to C. tibetica in the
characters of persistent rachis and full
of flosses inside and outside of the
pod, but different in the lobular involu-
tion, tubular, O-shaped cross-sec-
tion, etc. However, the ITS phylogenet-
ic tree showed that C. ordosica was
closely related to C. roborovskyi (BV=
100%).
In addition, ZHAO Yi-zhi[19] thought
that the scientific name of Zhongjian
jinjier should be C. davazamcii Sancz,
while C. intermedia Kuang et HC Fu,
C. erenensis Lion f., C. korshinskii
Kom. Var. Davazamcii (Schcz) Yakovl
and C. korshinskii Kom. Var. ordosiac
Yakovl were its synonyms. However,
ITS evidence suggested that C. dav-
azamcii was separated from other
species (Fig. 1), so the C. davazamcii,
C. korshinskii,C. intermedia, and C. ko-
rshinskii were not synonyms. These
results suggested that the studies on
species classification and the relation-
ships among species by the morpho-
38
Agricultural Science & Technology2012
Vol.13, No.1, 2012 Agricultural Science & Technology
利用 ITS序列探讨锦鸡儿属（Caragana Fabr.）植物系统关系
康红梅 1，2，白 娟 3，陈 康 4，王 刚 1* （1. 兰州大学生命科学院，甘肃兰州 730000；2. 甘肃省科学院生物研究所，甘肃兰州 730000；3. 西北农林
科技大学生命科学院, 陕西杨陵 712100；4.中国科学院海洋研究所，山东青岛 266071）
摘 要 [目的] 利用 ITS序列探讨锦鸡儿属（Caragana Fabr.）植物系统关系。[方法]以锦鸡儿属 11个系 29种为代表材料，选择性扩增 nrITS序列
并双向测序，结合黄耆亚族 Astralinae (Adens) Benth 其他 6属 7个代表种的 nrITS序列进行最大简约性（MP）和最小进化（ME）的系统发育分析。
[结果]锦鸡儿植物 ITS序列长度在 611-614 bp之间，与外类群排序后长度为 655 bp，共有 170个可变位点，其中 107个简约信息位点，简约信息
位点在总排序序列中达 16.3%，可以为属内及属间系统关系提供有力的分子证据；锦鸡儿属在系统发育上不是一个单系类群，与丽豆属
(Calophaca Fish. ex DC.) 植物具有极为相近的亲缘关系；Sect. tragacanthoides的种在 MP和 ME进化树中位置分散，其组的分类有待进一步研
究。卷叶锦鸡儿（C. ordosica，新种）虽然形态上与垫状锦鸡儿（C. tibetica）相似，但它与荒漠锦鸡儿（C. roborovskyi）遗传学关系紧密；C. davazamcii
是一个独立的种。[结论] ITS序列在锦鸡儿属内及属间的系统学研究中具有重要的参考价值。
关键词 锦鸡儿属；ITS序列；系统关系
基金项目 国家重点基础研究发展规划 (2002CB111505)。
作者简介 康红梅(1973- )，女，河南人，博士研究生，主要从事植物生态学及恢复生态学的研究工作，E-mail：lzkanghm@163.com。*通讯作者，博
士生导师。
收稿日期 2011-11-02 修回日期 2011-11-28
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
logical study was not enough.
Overall, ITS sequences have an
important reference value in exploring
the relationships of Caragana. ITS
analysis suggested that there were five
groups in this genus. However, there
were only 29 species in this study, so
more molecular evidence were needed
for the further study on the phylogenet-
ic relationships inCaragana.
Acknowledgements
Thanks for the help of LI Xin-rong,
ZHOU Hai-yan, ZHAO Jun-long from
Shapotou Workstation Introduction
Garden of Cold and Arid Regions En-
vironmental and Engineering Re-
search Institute, Chinese Academy of
Sciences and WANG Ji-he, WEI Qiu-
shi from Sand Plant Garden of Gansu
Desert Control Research Institute.
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Responsible editor: Tingting LI Responsible proofreader: Xiaoyan WU
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